Pyrimidine 5 -nucleotidases

Pyrimidine 5 -nucleotidase (P5N) is a unique enzyme that was recognized from studies of families with relatively common hemolytic disorders. The enzyme catalyzes the hydrolytic dephosphorylation of pyrimidine 5 -nucleotides but not purine nucleotides. The role of this enzyme is to eliminate RNA and DNA degradation products from the cytosol during erythroid maturation by conversion of nucleotide monophosphates to diffusible nucleosides. P5N is inhibited by lead, and its activity is considered to be a good indicator of lead exposure (PI). 

Fig. 6. Basophilic stippling in peripheral blood smear of a patient with pyrimidine 5 -nucleotidase deficiency.

Pyrimidine 5 -nucleotidase (P5N) deficiency appears to be the third most common cause of hereditary nonspherocytic hemolytic anemia after G6PD and PK deficiencies. To date, more than 42 cases have been reported worldwide (FI 1) since the first report by Valentine et al. (V4). This syndrome is characterized by hemolytic anemia, pronounced basophilic stippling of red blood cells (Fig. 6), and a... 

H6. Hirono, A., Fujii, H Natori, H., Kurokawa, I., and Miwa, S., Chromatographic analysis of human erythrocyte pyrimidine 5 -nucleotidase from five patients with primidine 5 -nucleotidase deficiency. Br. J. Haematol. 65,35-41 (1987). 

M26. Miwa, S., Luzzatto, L., Rosa, R., Paglia, D. E., Schroter, W De Flora, A., Fujii, H., Board, P. G and Beutler, E., International Committee for Standardization in Haematology Recommended methods for an additional red cell enzyme (pyrimidine 5 -nucleotidase) assay and the determination of red cell adenosine 5 -triphosphate, 2,3-diphosphoglycerate and reduced glutathione. Clin. Lab. Haematol. 11, 131-138 (1989). 

V4. Valentine, W. N., Fink, K Paglia, D. E., Harris, S. R., and Adams, W. S., Hereditary hemolytic anemia with human erythrocyte pyrimidine 5 -nucleotidase deficiency. J. Clin. Invest. 54, 866-879 (1974). 

ALA = 5-aminolevulinic acid ALAD = 6-aminolevulinic acid dehydratase ALAS = 5-aminolevulinic acid synthase EP = erythrocyte protoporphyrins FEP = free erythrocyte protoporphyrins FSH = follicle stimulating hormone IQ = intelligence quotient LH = luteinizing hormone NS = not specified (occup) = occupational Py-5 -N = pyrimidine-5-nucleotidase TSH = thyroid stimulating hormone ZPP = erythrocyte protoporphyrin... 

The increase in erythrocyte destruction may be due in part to inhibition by lead of pyrimidine-5 -nucleotidase, which results in an accumulation of pyrimidine nucleotides (cytidine and uridine phosphates) in the erythrocyte or reticulocyte. This enzyme inhibition and nucleotide accumulation affect erythrocyte membrane stability and survival by alteration of cellular energetic (Angle et al. 1982 EPA 1986a). Formation of the heme-containing cytochromes is inhibited in animals treated intraperitoneally or orally... 

Buc HA, Kaplan JC. 1978. Red-cell pyrimidine 5 -nucleotidase and lead poisoning. Clin Chim Acta 87 49-55. 

Paglia DE, Valentine WN, Dahigren JG. 1975. Effects of low-level lead exposure on pyrimidine 5 -nucleotidase and other erythrocyte enzymes Possible role of pyrimidine 5 -nucleotidase in the pathogenesis of lead-induced anemia. J Clin Invest 56 1164-1169. 

Tomokuni K, Ogata M. 1980. Comparative study of effects of lead on the activity of erythrocyte pyrimidine 5 -nucleotidase and delta-aminolevulinate dehydratase in vivo and in vitro. Arch Toxicol 45 197-201. 

Tomokuni K, Ogata M. 1980. [In-vitro effect of heavy metals on the activity of pyrimidine 5 nucleotidase and gamma amino levulinate dehydratase in the human erythrocyte], Jap J Ind Health 22 282- 283. (Japanese). 

We discuss two assays for the measurement of pyrimidine 5 -nucleotidase activity. In the first, as described by Sakai et al. (1982), a pyrimidine nucleoside 5 -phosphate is hydrolyzed to form the corresponding pyrimidine nucleoside. 

Figure 9.89 Pyrimidine 5 -nucleotidase assay in undialyzed human erythrocyte lysate obtained by 1 5 dilution of packed cells with deionized water, (a) Separation of 1 nmol of CMP, cytidine, and uridine as the standards. Separation of the assay mixture, containing 50 mM Tris-HCl, pH 7.5, 0.2 mM CMP, 1 mM MgCl2, 1 mM DTT, and 20 fiL of lysate in 0.5 mL of total volume (b) at time zero and (c) after 30 minutes of incubation (c). (From Amici et al., 1994.)...

Pyrimidine-5 -nucleotidases are a group of enzymes dephos-phorylating pyrimidine nucleotides to the corresponding nucleosides. The pyrimidine bases diffuse out of the erythrocyte and the phosphates are retained. Pyrimidine phosphates are present on ribosomes of erythroblasts and reticulocytes, but there are normally no pyrimidines in mature RBCs. Two cytoplasmic forms of the enzyme were identified in the erythrocyte, P5 N-1 and P5 N-2. These enzymes are encoded by different genes and have different molecular properties and substrate specificities. Since there are no known disorders associated with deficiency of P5 N-2, this enzyme will not be further discussed here. 

Pyrimidine-5 -nucleotidase effects the release of phosphate from cytidine-5 -monophosphate and uridine-5 -monophosphate and thus is an enzyme involved in the catabolism of RNA. RNA is a normal constituent of reticulocytes but not of mature RBCs. In the absence (or notable deficiency) of P-5 -N, RNA persists in RBCs and is manifested by striking basophilic stippling and mild to moderate hemolytic anemia. 

Balta G, Gumruk F, Akarsu N, Gurgey A, Altay C. Molecular characterization of Turkish patients with pyrimidine 5 nucleotidase-I deficiency. Blood 2003 102 1900-3. 

Bianchi P, Fermo E, Alfinito F, Vercellati C, Baserga M, Ferraro F, et al. Molecular characterization of six unrelated Italian patients affected by pyrimidine 5 -nucleotidase deficiency. Br J Haematol 2003 122 847-51. 

Marinaki AM, Escuredo E, Duley JA, Simmonds HA, Amici A, Naponelli V, et al. Genetic basis of hemolytic anemia caused by pyrimidine 5 nucleotidase deficiency. Blood 2001 97 3327-32. 

Rees DC, Duley JA, Marinaki AM. Pyrimidine 5 nucleotidase deficiency. Br J Haematol 2003 120 375-83. 

Swallow DM, Aziz I, Hopkinson DA, Miwa S. Analysis of human erythrocyte 5 -nucleotidases in healthy individuals and a patient deficient in pyrimidine 5 -nucleotidase. Ann Hum Genet 1983 47 Pt 1 19-23. 

A number of other enzymopathic substances (e.g., pyruvate kinase. Chapter 13 and pyrimidine-5 -nucleotidase. Chapter 27), abnormal hemoglobins (Chapter 28), and abnormalities of the erythrocyte cytoskeleton (Chapter 10) may cause hemolytic anemia. Because many enzymes in the red cell are identical to those in other tissues, defects in these enzymes may have pleiotropic effects. Thus, in addition to hemolytic anemia, triose phosphate isomerase deficiency causes severe neuromuscular disease, and phospho-fructokinase deficiency causes a muscle glycogen storage disease (Chapter 13). Mutations that result in decreased enzyme stability are usually most strongly expressed in erythrocytes because of their inability to synthesize proteins. 

DIAGNOSTIC AND THERAPEUTIC APPROACHES IN PYRIMIDINE 5 -NUCLEOTIDASE DEFICIENCY... 

Hereditary deficiency of erythrocyte pyrimidine 5 nucleotidase results in a chronic haemolytic anaemia. The red cells show basophilic stippling and contain a markedly increased content of nucleotides, 3-6 times greater than normal, and 65 to 80% of nucleotides are pyrimidine in type (Valentine, et al., 1974 Torrance and Whittaker, 1979). Pyrimidine 5 nucleotidase is unique amongst the 5 nucleotidases in its strict substrate specificity for pyrimidine nucleoside 5 monophosphates, its pH profile, and for its cytosolic localisation (Paglia and Valentine, 1975). 

---